Odorant receptors are members of the seven transmembrane receptor superfamily. The receptors are linked to second messenger systems by GTP- binding proteins (G-proteins). In vertebrates, odorants may stimulate adenylate cyclase to increase cyclic AMP levels or may stimulate phospholipase C to form two second messengers, inositol 1,4,5- trisphosphate and diacylglycerol. The adenylate cyclase/cyclic AMP signaling pathway has been extensively studied and the transduction proteins mediating this pathway in olfactory neurons have been identified. In contrast, the identities of the proteins mediating phosphoinositide signaling in olfaction have not been determined. The goal of the proposed research is to address some of the gaps in our understanding of phosphoinositide signaling in olfactory neurons by studying the molecular natures of two transduction proteins in catfish olfactory epithelium. The target transduction proteins are the odorant-sensitive phospholipase C (PLC) and the protein kinase C (PKC) mediating olfactory signal termination. The most likely candidate(s) for the odorant-sensitive PLC are members of the PLCbeta subfamily. These genes will be identified by screening cDNA libraries, PCR and immunocytochemistry. Since the activity of some members of the PLCbeta subfamily is regulated by G-protein beta- gamma subunits, the composition of the beta-gamma pool expressed in olfactory cilia will be studied by Western blotting, PCR and immunohistochemistry. Several PKC isotypes have been identified in catfish olfactory rosettes. One of these appears to represent a new PKC sequence. A cDNA clone encoding the unique PKC will be isolated from cDNA libraries. The clone will be expressed in SF9 cells using a baculovirus expression system and the biochemical properties of the recombinant protein will be determined. The expression of PKC isotypes in olfactory cilia will also be studied by immunohistochemistry. When candidate transduction proteins have been identified, additional experiments will be performed to establish functionality in olfactory transduction. Completion of the proposed identification experiments will provide the necessary data for the design and use of antisense oligonucleotides in vivo to block expression of individual signaling components.